Staphylococcus aureus is a major public health concern and is responsible for over 300,000 hospitalizations in the United States each year (1-2). The organism is a leading cause of suppurative skin lesions, bacterial endocarditis, bacteremia, and pneumonia. Treatment of S. aureus infection is complicated by the emergence of numerous antibiotic resistant strains (2). Once previously limited to hospital settings, community acquired infections due to S. aureus continue to arise, often in otherwise healthy individuals (2). S. aureus secretes an arsenal of virulence factors that promote the organism's survival in susceptible hosts. Among these factors are a number of secreted toxins that directly interact with and kill/damage host cells (3-5). The coordinated regulation of toxin expression is critical to S. aureus pathogenesis. Our data demonstrate that regulation of toxin production by transcriptional repression is critical to optimal expression of the leukotoxin LukED. When such regulatory patterns are perturbed, S. aureus strains become hypervirulent in mouse models of infection due to increased LukED production. Experiments described in this application are designed to (i) elucidate the regulatory mechanism(s) of lukED gene expression in S. aureus and (ii) determine the functional impact of LukED intoxication in vitro, ex vivo, and in vivo. Bacterial genetics, promoter binding assays, in vitro cell toxicity and survival assays, in vivo models of septicemic infection, and immunological techniques will be critical to the execution of the above aims. Experiments will investigate the impact of coordinated lukED gene expression in vivo, the role of the toxin in bacterial mediated mammalian cell killing, and the toxin's effecton immune cell recruitment/viability in vivo. Ultimately, we will determine the major contributions of LukED to pathogenesis in vivo and will provide insight into the optimal toxin expression patterns required for infection. Importantly, this work will further the mission of the National Institutes f Health by examining the mechanistic underpinnings of disease caused by S. aureus, with an end goal of improving prevention and treatment strategies. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus infections continue to be a major public health concern in the United States where they result in over 300,000 hospitalizations yearly. More than 1.3 million people are affected by S. aureus-related infections in any given year, further highlighting the need to investigate the underlying mechanisms that allow this organism to cause disease. This application will investigate the regulation and activity of an important toxin that promotes disease in S. aureus; such studies have the potential to determine the mechanism of action of a novel target for inhibitory drug design and vaccine therapy.